15 research outputs found
Computational Workflow for Refining AlphaFold Models in Drug Design Using Kinetic and Thermodynamic Binding Calculations: A Case Study for the Unresolved Inactive Human Adenosine A<sub>3</sub> Receptor
A structure-based
drug design pipeline that considers
both thermodynamic
and kinetic binding data of ligands against a receptor will enable
the computational design of improved drug molecules. For unresolved
GPCR-ligand complexes, a workflow that can apply both thermodynamic
and kinetic binding data in combination with alpha-fold (AF)-derived
or other homology models and experimentally resolved binding modes
of relevant ligands in GPCR-homologs needs to be tested. Here, as
test case, we studied a congeneric set of ligands that bind to a structurally
unresolved G protein-coupled receptor (GPCR), the inactive human adenosine
A3 receptor (hA3R). We tested three available
homology models from which two have been generated from experimental
structures of hA1R or hA2AR and one model was
a multistate alphafold 2 (AF2)-derived model. We applied alchemical
calculations with thermodynamic integration coupled with molecular
dynamics (TI/MD) simulations to calculate the experimental relative
binding free energies and residence time (Ï„)-random accelerated
MD (Ï„-RAMD) simulations to calculate the relative residence
times (RTs) for antagonists. While the TI/MD calculations produced,
for the three homology models, good Pearson correlation coefficients,
correspondingly, r = 0.74, 0.62, and 0.67 and mean
unsigned error (mue) values of 0.94, 1.31, and 0.81 kcal mol–1, the τ-RAMD method showed r = 0.92 and 0.52
for the first two models but failed to produce accurate results for
the multistate AF2-derived model. With subsequent optimization of
the AF2-derived model by reorientation of the side chain of R1735.34 located in the extracellular loop 2 (EL2) that blocked
ligand’s unbinding, the computational model showed r = 0.84 for kinetic data and improved performance for thermodynamic
data (r = 0.81, mue = 0.56 kcal mol–1). Overall, after refining the multistate AF2 model with physics-based
tools, we were able to show a strong correlation between predicted
and experimental ligand relative residence times and affinities, achieving
a level of accuracy comparable to an experimental structure. The computational
workflow used can be applied to other receptors, helping to rank candidate
drugs in a congeneric series and enabling the prioritization of leads
with stronger binding affinities and longer residence times
Dual A1/A3 Adenosine Receptor Antagonists: Binding Kinetics and Structure−Activity Relationship Studies Using Mutagenesis and Alchemical Binding Free Energy Calculations
Drugs targeting adenosine receptors
(AR) can provide treatment
for diseases. We report the identification of 7-(phenylamino)-pyrazolo[3,4-c]​pyridines L2–L10, A15, and A17 as low-micromolar to low-nanomolar A1R/A3R dual antagonists, with 3-phenyl-5-cyano-7-(trimethoxyphenylamino)-pyrazolo[3,4-c]​pyridine (A17) displaying the highest
affinity at both receptors with a long residence time of binding,
as determined using a NanoBRET-based assay. Two binding orientations
of A17 produce stable complexes inside the orthosteric
binding area of A1R in molecular dynamics (MD) simulations,
and we selected the most plausible orientation based on the agreement
with alanine mutagenesis supported by affinity experiments. Interestingly,
for drug design purposes, the mutation of L2506.51 to alanine
increased the binding affinity of A17 at A1R. We explored the structure–activity relationships against
A1R using alchemical binding free energy calculations with
the thermodynamic integration coupled with the MD simulation (TI/MD)
method, applied on the whole G-protein-coupled receptor–membrane
system, which showed a good agreement (r = 0.73)
between calculated and experimental relative binding free energies
Dual A1/A3 Adenosine Receptor Antagonists: Binding Kinetics and Structure−Activity Relationship Studies Using Mutagenesis and Alchemical Binding Free Energy Calculations
Drugs targeting adenosine receptors
(AR) can provide treatment
for diseases. We report the identification of 7-(phenylamino)-pyrazolo[3,4-c]​pyridines L2–L10, A15, and A17 as low-micromolar to low-nanomolar A1R/A3R dual antagonists, with 3-phenyl-5-cyano-7-(trimethoxyphenylamino)-pyrazolo[3,4-c]​pyridine (A17) displaying the highest
affinity at both receptors with a long residence time of binding,
as determined using a NanoBRET-based assay. Two binding orientations
of A17 produce stable complexes inside the orthosteric
binding area of A1R in molecular dynamics (MD) simulations,
and we selected the most plausible orientation based on the agreement
with alanine mutagenesis supported by affinity experiments. Interestingly,
for drug design purposes, the mutation of L2506.51 to alanine
increased the binding affinity of A17 at A1R. We explored the structure–activity relationships against
A1R using alchemical binding free energy calculations with
the thermodynamic integration coupled with the MD simulation (TI/MD)
method, applied on the whole G-protein-coupled receptor–membrane
system, which showed a good agreement (r = 0.73)
between calculated and experimental relative binding free energies
Dual A1/A3 Adenosine Receptor Antagonists: Binding Kinetics and Structure−Activity Relationship Studies Using Mutagenesis and Alchemical Binding Free Energy Calculations
Drugs targeting adenosine receptors
(AR) can provide treatment
for diseases. We report the identification of 7-(phenylamino)-pyrazolo[3,4-c]​pyridines L2–L10, A15, and A17 as low-micromolar to low-nanomolar A1R/A3R dual antagonists, with 3-phenyl-5-cyano-7-(trimethoxyphenylamino)-pyrazolo[3,4-c]​pyridine (A17) displaying the highest
affinity at both receptors with a long residence time of binding,
as determined using a NanoBRET-based assay. Two binding orientations
of A17 produce stable complexes inside the orthosteric
binding area of A1R in molecular dynamics (MD) simulations,
and we selected the most plausible orientation based on the agreement
with alanine mutagenesis supported by affinity experiments. Interestingly,
for drug design purposes, the mutation of L2506.51 to alanine
increased the binding affinity of A17 at A1R. We explored the structure–activity relationships against
A1R using alchemical binding free energy calculations with
the thermodynamic integration coupled with the MD simulation (TI/MD)
method, applied on the whole G-protein-coupled receptor–membrane
system, which showed a good agreement (r = 0.73)
between calculated and experimental relative binding free energies
Dual A1/A3 Adenosine Receptor Antagonists: Binding Kinetics and Structure−Activity Relationship Studies Using Mutagenesis and Alchemical Binding Free Energy Calculations
Drugs targeting adenosine receptors
(AR) can provide treatment
for diseases. We report the identification of 7-(phenylamino)-pyrazolo[3,4-c]​pyridines L2–L10, A15, and A17 as low-micromolar to low-nanomolar A1R/A3R dual antagonists, with 3-phenyl-5-cyano-7-(trimethoxyphenylamino)-pyrazolo[3,4-c]​pyridine (A17) displaying the highest
affinity at both receptors with a long residence time of binding,
as determined using a NanoBRET-based assay. Two binding orientations
of A17 produce stable complexes inside the orthosteric
binding area of A1R in molecular dynamics (MD) simulations,
and we selected the most plausible orientation based on the agreement
with alanine mutagenesis supported by affinity experiments. Interestingly,
for drug design purposes, the mutation of L2506.51 to alanine
increased the binding affinity of A17 at A1R. We explored the structure–activity relationships against
A1R using alchemical binding free energy calculations with
the thermodynamic integration coupled with the MD simulation (TI/MD)
method, applied on the whole G-protein-coupled receptor–membrane
system, which showed a good agreement (r = 0.73)
between calculated and experimental relative binding free energies
Dual A1/A3 Adenosine Receptor Antagonists: Binding Kinetics and Structure−Activity Relationship Studies Using Mutagenesis and Alchemical Binding Free Energy Calculations
Drugs targeting adenosine receptors
(AR) can provide treatment
for diseases. We report the identification of 7-(phenylamino)-pyrazolo[3,4-c]​pyridines L2–L10, A15, and A17 as low-micromolar to low-nanomolar A1R/A3R dual antagonists, with 3-phenyl-5-cyano-7-(trimethoxyphenylamino)-pyrazolo[3,4-c]​pyridine (A17) displaying the highest
affinity at both receptors with a long residence time of binding,
as determined using a NanoBRET-based assay. Two binding orientations
of A17 produce stable complexes inside the orthosteric
binding area of A1R in molecular dynamics (MD) simulations,
and we selected the most plausible orientation based on the agreement
with alanine mutagenesis supported by affinity experiments. Interestingly,
for drug design purposes, the mutation of L2506.51 to alanine
increased the binding affinity of A17 at A1R. We explored the structure–activity relationships against
A1R using alchemical binding free energy calculations with
the thermodynamic integration coupled with the MD simulation (TI/MD)
method, applied on the whole G-protein-coupled receptor–membrane
system, which showed a good agreement (r = 0.73)
between calculated and experimental relative binding free energies
Dual A1/A3 Adenosine Receptor Antagonists: Binding Kinetics and Structure−Activity Relationship Studies Using Mutagenesis and Alchemical Binding Free Energy Calculations
Drugs targeting adenosine receptors
(AR) can provide treatment
for diseases. We report the identification of 7-(phenylamino)-pyrazolo[3,4-c]​pyridines L2–L10, A15, and A17 as low-micromolar to low-nanomolar A1R/A3R dual antagonists, with 3-phenyl-5-cyano-7-(trimethoxyphenylamino)-pyrazolo[3,4-c]​pyridine (A17) displaying the highest
affinity at both receptors with a long residence time of binding,
as determined using a NanoBRET-based assay. Two binding orientations
of A17 produce stable complexes inside the orthosteric
binding area of A1R in molecular dynamics (MD) simulations,
and we selected the most plausible orientation based on the agreement
with alanine mutagenesis supported by affinity experiments. Interestingly,
for drug design purposes, the mutation of L2506.51 to alanine
increased the binding affinity of A17 at A1R. We explored the structure–activity relationships against
A1R using alchemical binding free energy calculations with
the thermodynamic integration coupled with the MD simulation (TI/MD)
method, applied on the whole G-protein-coupled receptor–membrane
system, which showed a good agreement (r = 0.73)
between calculated and experimental relative binding free energies
Dual A1/A3 Adenosine Receptor Antagonists: Binding Kinetics and Structure−Activity Relationship Studies Using Mutagenesis and Alchemical Binding Free Energy Calculations
Drugs targeting adenosine receptors
(AR) can provide treatment
for diseases. We report the identification of 7-(phenylamino)-pyrazolo[3,4-c]​pyridines L2–L10, A15, and A17 as low-micromolar to low-nanomolar A1R/A3R dual antagonists, with 3-phenyl-5-cyano-7-(trimethoxyphenylamino)-pyrazolo[3,4-c]​pyridine (A17) displaying the highest
affinity at both receptors with a long residence time of binding,
as determined using a NanoBRET-based assay. Two binding orientations
of A17 produce stable complexes inside the orthosteric
binding area of A1R in molecular dynamics (MD) simulations,
and we selected the most plausible orientation based on the agreement
with alanine mutagenesis supported by affinity experiments. Interestingly,
for drug design purposes, the mutation of L2506.51 to alanine
increased the binding affinity of A17 at A1R. We explored the structure–activity relationships against
A1R using alchemical binding free energy calculations with
the thermodynamic integration coupled with the MD simulation (TI/MD)
method, applied on the whole G-protein-coupled receptor–membrane
system, which showed a good agreement (r = 0.73)
between calculated and experimental relative binding free energies
Dual A1/A3 Adenosine Receptor Antagonists: Binding Kinetics and Structure−Activity Relationship Studies Using Mutagenesis and Alchemical Binding Free Energy Calculations
Drugs targeting adenosine receptors
(AR) can provide treatment
for diseases. We report the identification of 7-(phenylamino)-pyrazolo[3,4-c]​pyridines L2–L10, A15, and A17 as low-micromolar to low-nanomolar A1R/A3R dual antagonists, with 3-phenyl-5-cyano-7-(trimethoxyphenylamino)-pyrazolo[3,4-c]​pyridine (A17) displaying the highest
affinity at both receptors with a long residence time of binding,
as determined using a NanoBRET-based assay. Two binding orientations
of A17 produce stable complexes inside the orthosteric
binding area of A1R in molecular dynamics (MD) simulations,
and we selected the most plausible orientation based on the agreement
with alanine mutagenesis supported by affinity experiments. Interestingly,
for drug design purposes, the mutation of L2506.51 to alanine
increased the binding affinity of A17 at A1R. We explored the structure–activity relationships against
A1R using alchemical binding free energy calculations with
the thermodynamic integration coupled with the MD simulation (TI/MD)
method, applied on the whole G-protein-coupled receptor–membrane
system, which showed a good agreement (r = 0.73)
between calculated and experimental relative binding free energies
Dual A1/A3 Adenosine Receptor Antagonists: Binding Kinetics and Structure−Activity Relationship Studies Using Mutagenesis and Alchemical Binding Free Energy Calculations
Drugs targeting adenosine receptors
(AR) can provide treatment
for diseases. We report the identification of 7-(phenylamino)-pyrazolo[3,4-c]​pyridines L2–L10, A15, and A17 as low-micromolar to low-nanomolar A1R/A3R dual antagonists, with 3-phenyl-5-cyano-7-(trimethoxyphenylamino)-pyrazolo[3,4-c]​pyridine (A17) displaying the highest
affinity at both receptors with a long residence time of binding,
as determined using a NanoBRET-based assay. Two binding orientations
of A17 produce stable complexes inside the orthosteric
binding area of A1R in molecular dynamics (MD) simulations,
and we selected the most plausible orientation based on the agreement
with alanine mutagenesis supported by affinity experiments. Interestingly,
for drug design purposes, the mutation of L2506.51 to alanine
increased the binding affinity of A17 at A1R. We explored the structure–activity relationships against
A1R using alchemical binding free energy calculations with
the thermodynamic integration coupled with the MD simulation (TI/MD)
method, applied on the whole G-protein-coupled receptor–membrane
system, which showed a good agreement (r = 0.73)
between calculated and experimental relative binding free energies